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WO2002088351A1 - Cellules et organes modifies destines a la xenotransplantation - Google Patents

Cellules et organes modifies destines a la xenotransplantation Download PDF

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WO2002088351A1
WO2002088351A1 PCT/US2002/013564 US0213564W WO02088351A1 WO 2002088351 A1 WO2002088351 A1 WO 2002088351A1 US 0213564 W US0213564 W US 0213564W WO 02088351 A1 WO02088351 A1 WO 02088351A1
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cells
human
gal
epitopes
prbcs
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Alex Zhu
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RBC Biotechnology Inc
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RBC Biotechnology Inc
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Priority to ES02766869T priority patent/ES2530872T3/es
Priority to EP02766869.8A priority patent/EP1383889B1/fr
Priority to CA002445945A priority patent/CA2445945A1/fr
Priority to JP2002585632A priority patent/JP2004533242A/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

  • the present invention is generally in the field of xenotransplantation, and genetic modification of animals to produce tissue, cells or organs less likely to induce rejection following transplantation. Shortage of Organs for Transplantation
  • organ allografts i.e., transplants from one animal into another animal of the same species, such as human to human
  • organ allografts are a routine treatment option for end-stage kidney, heart, lung, liver and other organ disease.
  • Xenografts i.e., transplants from one animal into another animal of a different species, such as from a pig into a human
  • xenografts will be developed using genetic engineering of non-primate species that are suitable for long-term replacement of damaged or diseased organs and subject only to minimal rejection.
  • the organs are still useful even if subject to some form of rejection by the new host.
  • rejection frequently develops and so patients are immunosuppressed using drugs such as cyclosporine and other types of immunosuppressants to prevent rejection of the allograft.
  • drugs such as cyclosporine and other types of immunosuppressants to prevent rejection of the allograft.
  • One solution to the problem of organ supply would be the use of organs taken from a suitable animal donor. Although the higher nonhuman primates (apes and Old World monkeys) would provide the closest immunological match for humans, there are several factors that make the routine use of these species as organ donors unlikely.
  • Xenotransplants between closely-related species can usually survive the initial period of blood perfusion without damage, as do allotransplants. Subsequently, the foreign antigens of the transplanted organ trigger the recipient's immune response and the rejection process begins. These xenografts, which are rejected clinically rather like allografts, but in an accelerated manner, are termed concordant xenotransplants.
  • Xenografts between phylogenetically more distant species e.g., pig-to-human
  • antibody-mediated (vascular) rejection generally occurs within a few minutes or hours of recirculation, with a typical histopathological pattern of endothelial lesions with severe interstitial hemorrhage and edema.
  • This hyperacute rejection is usually irreversible, but can be delayed by removal of the recipient's natural antibodies against the donor tissue.
  • This hyperacute rejection is entirely or largely a result of antibody-mediated complement activation through the classical pathway, as reported by Paul, L.C in Xenotransplantation Heidelberg, Springer, 1991. pp. 47-67; and Platt, J.L., Bach, F.H. In Xenotransplantation, Heidelberg, Springer, 1991.
  • Gal epitopes on the animal tissues by inserting a gene for an enzyme that competes with ⁇ l,3GT for the common substrate, N- acetyllactosamine, thus reducing the immune response following transplantation.
  • the DNA encoding another enzyme for modification of the sugar structures such as a sialyltransferase or a fucosyltransferase, can be inserted into the embryo where it is incorporated into the animal's chromosomes and expressed to modify or reduce the irnrnunoreactivity of the Gal structures on the cell surfaces.
  • mice This has been achieved in mice (Osman, N., et al, Proc. Natl. Acad. Sci. USA. 94, 14677-14682, 1997; Shinkel, T.A., et al. Transplantation 64. 197- , 1997; Tanemura, M., et al. Transplant. Proc. 29, 895, 1997) but to date has been only partially successful in pigs (Koike, C, et al, Xenotransplantation 3, 81-86, 1996; Sharma, A., et al. Proc. Natl. Acad. Sci. USA 93, 7190-7195, 1996), and has been reviewed by Cooper, D.K.C. Xenotransplantation 5. 6-17, 1998.
  • the epitope it is preferable to modify the epitope to a carbohydrate that is present in the human subject so that antibodies against this carbohydrate are not present in the human recipient of the animal organ. If it is modified to any other carbohydrate, then antibodies to this carbohydrate might develop if the carbohydrate is not naturally occurring in the human subject.
  • This may be achieved by genetically engineering the animals which serve as the source of the xenografts to express either a sialyltransferase or fucosyltransferase so that nonGal carbohydrate structures (that are also present in humans) are attached to the substrate (which is usually used for the formation of Gal epitopes) to prevent recognition and binding by the naturally occurring anti- Gal antibodies (Osman, N., et al, J. Biol. Chem. 271, 33105-33109, 1996; Osman, N., et al. Proc. Natl. Acad. Sci. USA. 94, 14677-14682, 1997;
  • Anti-Gal antibodies can be removed from human plasma by plasma exchange or adsorbed by passing the plasma through an immunoaffinity column of one or more of the specific Gal structures.
  • Gal carbohydrate The adsorption of such anti-pig antibodies by the specific Gal carbohydrate can prevent the hyperacute rejection that occurs when xenotransplantation is carried out between pig and a nonhuman primate, as reported by Ye, Y., et al. Transplantation. 58, 330-337, 1994; by Cooper, D.K.C., et al. Xeno. 4, 27-29, 1996; and by Xu, Y., et al. Transplantation. 65, 172-179, 1998.
  • Gal carbohydrates e.g., synthetic Gal oligosaccharides
  • Gal and NeuGc specific sugars
  • Gal epitope for example, Gal ⁇ (l- >3)Gal ⁇ (l->4)GlcNAc (linear B type 2), Gal ⁇ (l->3)Gal ⁇ (l->4)Glc (linear B type 6), Gal ⁇ (l->3)Gal (B disaccharide), and Gala (a-D-galactose).
  • the second is an N-glycolylneuraminic acid (NeuGc) structure.
  • NeuGc or the NeuGc and Gal epitopes, are either not produced or their expression is greatly reduced, or by chemical or enzymatic treatment of its cells to remove the epitopes, it is possible to produce organs, tissues and cells suitable for xenotransplantation into humans.
  • cells can be rendered even more compatible by genetically engineering the animal cells to express one or more human complement regulatory proteins, such as decay accelerating factor (DAF), membrane cofactor protein (MCP), or CD59 (as described by Cozzi E & White DJG. Nature Med 1: 964-966, 1995; and by Dalmasso AP, et al. Transplantation 1991;52: 530-3), or to express an excess of one or more porcine complement regulatory proteins (as reported by van den Berg CW & Morgan BP. Graft 4, 63-65, 2001).
  • DAF decay accelerating factor
  • MCP membrane cofactor protein
  • CD59 as described by Cozzi E & White DJG. Nature Med 1: 964-966, 1995; and by Dalmasso AP, et al. Transplantation 1991;52: 530-3
  • porcine complement regulatory proteins as reported by van den Berg CW & Morgan BP. Graft 4, 63-65, 2001.
  • Animals or cells can be engineered or treated enzymatically in vitro to remove the Gal epitopes and NeuGc epitopes, and in the most preferred method, to replace the NeuGc epitopes with NeuAc, thereby protecting them from destruction by antibody directed against the carbohydrate epitopes and yet maintaining their structural integrity and viability.
  • the same result can be achieved by developing genetically engineered animals, in particular pigs, by nuclear transfer technology or other genetic engineering, that do not express the Gal or NeuGc carbohydrate epitopes.
  • glycosylated recombinant therapeutic proteins can also be used to treat glycosylated recombinant therapeutic proteins to prevent their early antibody-mediated destruction when administered to humans.
  • the enzymatic treatment of the glycosylated recombinant proteins will prevent both injury from preformed antibody and the development of induced antibody in the human recipient directed against the carbohydrate epitopes (Gal and NeuGc) on the recombinant proteins.
  • these recombinant proteins can be derived from animals that have been genetically engineered not to express Gal or NeuGc.
  • Figure 1 is a schematic comparison of CMP-NeuAc hydroxylase cDNA from mouse (full length) and pig (partial sequence).
  • Figure 2 is a schematic of the partial sequence and gene structure for porcine CMP-NeuAc hydroxylase.
  • the three DNA fragments were amplified using the long PCR procedure from lambda DNA #46.
  • the grey areas are introns and the slashed areas are known sequences.
  • Three overlapped fragments, 3 kb, 5 kb, and 9kb, were obtained using both universal primers (SP6 and T7) and specific primers. Based on the sequence data, there are at least three introns in the coding regions.
  • Figure 3 is a schematic of the sequenced regions and gene structure for porcine CMP-NeuAc hydroxylase.
  • Region A (1614 bp), Region B (1268 bp), and Region C (2763 bp) sequences are shown in SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, respectively.
  • the single line represents unsequenced regions. Shaded areas, 1 through 5, represent exons with a total of 224 residues.
  • the sequences corresponding to the shaded areas, 1 through 5 are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.
  • Figure 4 is an amino acid sequence comparison between pig and chimpanzee CMP-NeuAc Hydroxylase.
  • the pig CMP-NeuAc hydozylase (SEQ ID NOs:4-8 combined) is compared with the chimpanzee enzyme (SEQ ID NO:9).
  • CMP-NeuAc hydroxylase cytidine monophospho-N-acetylneuraminic acid hydroxylase
  • ES cells embryonic stem cells
  • Gal the Gala l->3 Gal epitope
  • NeuAc N-acetylneuraminic acid
  • NeuGc N-glycolylneuraminic acid
  • N- acetylneuraminic acid (NeuAc), which is ubiquitously present in nature, and N-glycolylneuraminic acid (NeuGc), which is present in most animals with the notable exception of humans and chickens (Gottschalk, A. Glycoproteins: Their Composition. Structure and Function. Amsterdam. Elsevier, 1972; Asaoka, H., & Matsuda, H. J. Vet.Med. Sci. 56, 375-377, 1994).
  • NeuGc is synthesized in vivo from NeuAc by the addition of a single hydroxyi group by an enzyme called CMP-NeuAc hydroxylase (Shaw, L., Schauer, R. Biochem J 263: 355-363, 1989).
  • the gene encoding the enzyme has been cloned from mouse, chimpanzee and human (Kawano, T., et al. J. Biol. Chem. 270, 16458-16463, 1995). While mouse and chimpanzee genes code for a functional enzyme, the human gene has a partial deletion, resulting in a truncated form without enzymatic activity (Irie, A., & Suzuki, A. Biochem. Biophvs. Res.Commun.
  • anti-NeuGc antibodies antibodies against NeuGc
  • flow cytometry anti-NeuGc antibody was detected in most healthy human subjects tested, although the amount of it in the serum varied considerably.
  • Anti-NeuGc antibody activity can be specifically inhibited by pre-incubation with NeuGc molecules, but not with NeuAc molecules.
  • the identification of naturally-occuring anti-NeuGc antibody in healthy humans and the presence of NeuGc on pig vascular endothelial cells (Bouhours, D., et al. Glvcoconiugate J.
  • animals engineered to have the same altered glycosylation have utility as potential donors of organs, tissues or cells to humans.
  • Animals suitable for genetic engineering experiments can be obtained from standard commercial sources. These include animals such as mice and rats for testing of genetic manipulation procedures, as well as larger animals such as pigs, cows, sheep, goats, and other animals that have been genetically engineered using techniques known to those skilled in the art. These techniques are briefly summarized below based principally on manipulation of mice and rats .
  • mice Female animals are induced to superovulate using methodology adapted from the standard techniques used with mice, that is, with an injection of pregnant mare serum gonadotrophin (PMSG; Sigma) followed 48 hours later by an injection of human chorionic gonadotrophin (hCG; Sigma).
  • Females are placed with males immediately after hCG injection. Approximately one day after hCG, the mated females are sacrificed and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cells are removed with hyaluronidase (1 mg/ml). Pronuclear embryos are then washed and placed in Earle's balanced salt solution containing 0.5% BSA (EBSS) in a 37.5°C incubator with a humidified atmosphere at 5% CO 2 , 95% air until the time of injection.
  • EBSS Earle's balanced salt solution containing 0.5% BSA
  • Embryonic Stem (ESI Cell Methods; Nuclear Transfer and Cloning Methods for the culturing of cells and the subsequent production of genetically engineered animals, the introduction of DNA into cells by a variety of methods such as electroporation, calcium phosphate/DNA precipitation, and direct injection are described in detail in Teratocarcinomas and Embryonic Stem Cells, a Practical Approach, ed. E.J. Robertson, (IRL Press, 1987). Cloned pigs have also been produced using an updated nuclear transfer technology as reported by Polejaeva et al. (Nature. 407(6800):27, 29-30, 2000).
  • ES embryonic stem
  • a nucleic acid sequence for recombination with the ⁇ l,3GT gene or the gene for CMP-NeuAc hydroxylase, or sequences for controlling expression thereof is co-precipitated with a gene encoding a marker such as neomycin resistance.
  • Transfection is carried out by one of several methods described in detail in Lovell-Badge, in Teratocarcinomas and Embryonic Stem Cells, a Practical Approach, ed. E.J. Robertson, (IRL Press, 1987) or in Potter, H., et al. Proc. Natl. Acad. Sci. USA 81, 7161, 1984.
  • Calcium phosphate/DNA precipitation, direct injection, and electroporation are the preferred methods.
  • a number of cells for example, 0.5 X 10 6 , are plated into tissue culture dishes and transfected with a mixture of the linearized nucleic acid sequence and 1 mg of pSV2neo DNA (Southern, P.J., Berg, P. J. Mol. Appl. Gen. 1:327-341, 1982) precipitated in the presence of 50 mg lipofectin in a final volume of 100 ⁇ l.
  • the cells are fed with selection medium containing 10% fetal bovine serum in DMEM supplemented with an antibiotic such as G418 (between 200 and 500 ⁇ g/ml).
  • Colonies of cells resistant to G418 are isolated using cloning rings and expanded. DNA is extracted from drug resistant clones and Southern blotting experiments using the nucleic acid sequence as a probe are used to identify those clones carrying the desired nucleic acid sequences. In some experiments, PCR methods are used to identify the clones of interest.
  • DNA molecules introduced into cells can also be integrated into the chromosome through the process of homologous recombination, described by Capecchi, M.R. (Science. 244. 1288-1292, 1989). Direct injection results in a high efficiency of integration. Desired clones are identified through PCR of DNA prepared from pools of injected ES cells. Positive cells within the pools are identified by PCR subsequent to cell cloning (Zimmer, A., Grass, P. Nature 338, 150-153, 1989). DNA introduction by electroporation is less efficient and requires a selection step.
  • the recipient females are anesthetized.
  • the ovaries are exposed by making an incision in the body wall directly over the oviduct and the ovary and uterus are externalized.
  • a hole is made in the uterine horn with a needle through which the blastocysts are transferred.
  • the ovary and uterus are pushed back into the body and the incision is closed by suturing. This procedure is repeated on the opposite side if additional transfers are to be made.
  • Samples (1-2 cm of mouse tails) are removed from young animals. For larger animals, blood or other tissue can be used. To test for chimeras in the homologous recombination experiments, i.e., to look for contributions of the targeted ES cells to the animals, coat color has been used in mice, although blood could be examined in larger animals.
  • DNA is prepared and analyzed by both Southern blot and PCR to detect transgenic founder (F 0 ) animals and their progeny (Fj and F 2 ). Once the genetically-engineered animals are identified, lines are established by conventional breeding and used as the donors for tissue removal and implantation using standard techniques for implantation into humans.
  • DNA into the embryo using microinjection or other techniques known to those skilled in the art such as electroporation.
  • the DNA is selected on the basis of the purpose for which it is intended: to inactivate the gene encoding an enzyme such as the CMP-NeuAc hydroxylase or ⁇ l,3GT.
  • the enzyme- encoding gene can be modified by homologous recombination with a DNA for a defective enzyme, such as one containing within the coding sequence an antibiotic marker, which can then be used for selection purposes.
  • a knockout pig can be produced by the use of nuclear transfer technology (Colman A . Cloning. 1: 185-200, 2000; Polejaeva I, et al., Nature. 407 (6800), 27, 29-30, 2000).
  • a targeted disruption of the ⁇ l,3GT gene in porcine cells has been carried out (Polejaeva I, et al., Nature. 407 (6800) 27, 29-30, 2000).
  • the approach requires the knockout of the gene for ⁇ l,3GT in a single adult, fetal or embryonic cell, e.g., a fibroblast, and nuclear transfer of this modified cell using state-of-the-art techiques. This will result in the birth of a pig heterozygous for ⁇ l,3GT gene.
  • a preferred solution to the provision of pRBCs, cells, tissues and organs that are not targets for human natural antibodies would be the breeding of pigs that express neither Gal nor NeuGc epitopes (double- knockout pigs).
  • double- knockout pigs The cross-breeding of an ⁇ l,3GT-knockout pig with a CMP-NeuAc hydroxylase-knockout pig will produce a double-knockout pig.
  • the RBCs or any other cells or organs from the double-knockout pig will be deprived of the two major antigens against which humans have natural xenoreactive antibodies, Gal and NeuGc (and can therefore be characterized by such phenotype).
  • pig organs, tissues, cells or pRBCs should negate the need for other genetic manipulations of the pig, such as the creation of a pig transgenic for a human (or additonal pig) complement regulatory protein.
  • transgenic or nuclear transfer techniques will allow this additional protection of the pRBCs.
  • CD46 also known as membrane cofactor protein (MCP), as described by Purcell, D.F., et al., J. Immunol. 70:155-161, 1990; Lublin, D.M., et al. J.Exp.Med. 168. 181-194, 1988; and Seya, T. & Atkinson, J.P. Biochem. 1 264:581-588, 1989.
  • MCP membrane cofactor protein
  • This inhibitor functions by binding to complement component C3b thereby activating molecules that cleave C3b into inactive fragments preventing accumulation of C3b and, therefore, its contribution to the formation of the membrane attack complex (MAC).
  • MAC membrane attack complex
  • CD55 also known as decay accelerating factor (DAF) (described by Nicholson-Weller, A., et al., J. Immunol. 129:184-189, 1982; Lublin, D.M. & Atkinson, J.P. Annu. Rev. Immunol. 7:35-58, 1989; Medof, M.E. & Atkinson, J.P. J. Exp. Med. 165:1731-1736. 1987; and Medof. M.E., et al., Proc. Natl. Acad. Sci. USA 84:2007-2011. 1987).
  • DAF decay accelerating factor
  • This inhibitor is a membrane-bound protein of approximately 70 kD in molecular mass which interferes with the assembly of C3 convertase.
  • Cells suitable for transplantation into a foreign host are protected from complement-mediated lysis by introducing into the cell DNA encoding a protein, or combination of proteins, inhibiting complement-mediated lysis, for example, CD59, CD55, CD46 and or other inhibitors of C8 or C9 or certain other proteins in the complement cascade.
  • the DNA is introduced into the cells by transfection or infection with a vector encoding the complement- regulatory protein, and expressed on the surface of the transfected/infected cells.
  • the inhibitor is preferably of the same species of origin as the host into which the cells are to be transplanted.
  • the gene encoding the complement regulatory protein (inhibitor) can be introduced into a cell of a different species of origin (Cozzi, E., et al.
  • a human CD59 gene can be introduced into a porcine cell so that the cell resists attack when transplanted into a human, or the gene can be introduced into a cell of the same species of origin so that increased amounts of the protein are expressed on the surface of the cell (Nan den Berg CW, Morgan BP.Graft 4, 63-65, 2001).
  • the gene can be placed under the control of a promoter enhancing expression of the gene which is then inserted by homologous recombination into the host cell chromosome at the site where the gene is normally located, but under the control of the promoter which enhances expression, or can be inserted into the chromosome at another locus on the chromosome.
  • DNA encoding the complement inhibitors can be introduced into cells in culture using transfection or into embryos for production of transgenic animals expressing the complement inhibitors on the surface of their cells.
  • transfection can be accomplished by electroporation, calcium phosphate precipitation, a lipofectin-based procedure, or microinjection or through use of a "gene gun".
  • cDNA for the inhibitory protein, such as CD59 is subcloned into a plasmid-based vector which encodes elements for efficient expression in the genetically-engineered cell.
  • the plasmid-based vector preferably contains a marker such as the neomycin gene for selection of stable transfectants with the cytotoxic aminoglycoside G418 in eukaryotic cells and an ampicillin gene for plasmid selection in bacteria.
  • Infection is accomplished by incorporating the genetic sequence for the inhibitory protein into a retroviral vector.
  • Various procedures are known in the art for such incorporation.
  • One such procedure which has been widely used in the art employs a defective murine retrovirus, Psi-2 cells for packaging the retrovirus, and the amphotropic packaging cell line Psi-AM to prepare infectious amphotropic virus for use in infecting the target donor cells, as described by Kohn, D.B., et al., Blood Cells 13:285-298, 1987.
  • a retrovirus of the self-inactivating and double-copy type can be used, such as that described by Hantzopoulos, P.A., et al., Proc. Natl. Acad. Sci. USA 86:3519-3523, 1989. Enzymatic Modification of Xenoantigens on Cells
  • the Gal epitopes on cells such as porcine RBCs can be readily removed by treatment with ⁇ -galactosidase, resulting in RBCs that are no longer reactive with human natural anti-Gal antibody.
  • the other major carbohydrate xenoantigen on pRBCs, the NeuGc epitope can also be removed from the surface of pRBCs in vitro by the enzyme, neuraminidase.
  • this enzyme also removes NeuAc, which may lead to a reduction in the half-life of the cells, if transfused into humans in vivo.
  • This problem can be resolved by treating the neuraminidase-treated pRBCs with the enzyme, sialyltransferase, using CMP-NeuAc as a substrate.
  • the resulting pRBCs will thus have NeuAc, but not NeuGc, on the cell surface.
  • the same technology can be used to treat recombinant therapeutic proteins to make them more compatible with, and acceptable to, the human immune system.
  • Most therapeutic proteins are produced in non-human expression systems (mammalian cell lines and transgenic animals) and, if they are glycosylated, almost certainly express Gal and NeuGc epitopes.
  • analysis of a human anti-lipopolysaccharide IgM produced by a human-mouse heterohybridoma revealed the presence of Gal epitopes and high amounts of NeuGc; the ratio of NeuGc to NeuAc was found to be 98:2 (Leibiger, H., et al, Glycobiology.
  • ACTAVASE® plasminogen activator by Genentech, Inc.
  • ACTAVASE® is a recombinant protein used in patients undergoing myocardial infarction or cerebrovascular occlusion. Due to its short half-life (approximately 5 min), a large dose of the drug (approximately 100 mg) is required for therapeutic effect.
  • the protein is produced in a mammalian cell line (Chinese hamster ovary (CHO) cells). The presence of Gal and NeuGc in hamster cells almost certainly provides an explanation for the observed short half-life of the drug.
  • the technology described herein provides a means to increase the in vivo half-life.
  • the animal that is to provide the source of the cell line or in which the therapeutic protein (e.g., monoclonal antibody) is to be produced would be genetically engineered to be a Gal/NeuGc double knockout to avoid expression of these carbohydrates.
  • Example 1 Preparation of pig RBCs for xenotransplantation. Physiology of Pig Red Blood Cells
  • pRBCs share a number of common characteristics with human RBCs (Table 1) (Pond WG, Houpt KA. The Biology of the Pig. Ithaca: Comstock Pub. Associates, 1978; Jandl JH. Blood :Textbook of Hematologv. Boston: Little, Brown, 1996).
  • the pRBC is a biconcave disk of approximately 4-8 microns in diameter.
  • the hematocrit of pig blood is 35-47%, with a hemoglobin concentration of 6-17g/100ml.
  • the half-life of pRBC is approximately 40 days, in comparison to 60 days for human RBCs.
  • Table 1 Comparison of selected parameters relating to blood between pig and human.
  • A-O(H) system which is closely related to the human ABO system.
  • the A and O antigens on pRBC are passively adsorbed from circulating plasma glycosphingolipids, in a similar mechanism as human Lewis antigens (Marcus, D.M., Cass, L.E. Science 164: 553-555, 1969). pRBC phenotyping is therefore not entirely reliable.
  • Phenotyping of pigs can be achieved by immunohistochemical staining of buccal epithelial cells with an anti-A monoclonal antibody (mAb) (as used in Blood Banks) and an anti-H lectin antibody (Ulex europaeus) (Villarroya, H., et al, Autoimmunitv 6: 47-60, 1990).
  • mAb monoclonal antibody
  • Ulex europaeus Ulex europaeus
  • the glycolipids bearing blood group A have been isolated from porcine stomach mucosa (Slomiany, A., et al., J Biol Chem 249: 1225-1230, 1974), epithelial cells (Backer, A.E., et al.
  • porcine hemoglobin not only shares 85% sequence identity with its human counterpart but also demonstrates a similar three-dimensional structure at 2.8 A resolution (Katz, D.S., et al. J Mol Biol 244: 541-553, 1994). Furthermore, human hemoglobin has been expressed in transgenic pigs, with normal post-translational modifications and biological function (Rao, M.J., et al., Artif Cells Blood Substit Immobil Biotechnol 22: 695-700, 1994).
  • Another cloned protein from pRBC is the complement regulatory protein CD59 (Van Den Berg, C.W., et al., J. Immunol Methods 179: 223-231, 1995). Although the protein is only 48% identical to human CD59 at the amino acid level, pig CD59 is capable of regulating human complement activation (Hinchliffe, S.J., et al. J Immunol 160: 3924-3932, 1998).
  • A-like pRBCs into a human subject would result in cell lysis or agglutination of the pRBCs if the recipient were of blood type O or B (and therefore had preformed anti-A antibodies).
  • Pig herds are available, however, which have been bred to homogeneity for the O-like blood type, and therefore this potential problem can readily be avoided. All pRBCs to be therapeutically transfused into humans would be derived from O-like pigs.
  • pRBCs can, however, be treated in vitro with the enzyme ⁇ -galactosidase, which removes the terminal Gal sugar molecule from the surface of the cell, rendering the cell no longer susceptible to binding by anti-Gal antibodies (LaVecchio JA, et al., Transplantation 60: 841-847, 1995). Transfusion of pRBCs treated in this way would prevent lysis of the cells from this mechanism.
  • pRBCs and other tissues have other terminal sugar molecules expressed on their surface (Cooper, D.K.C. Xenotransplantation. 5, 6-17.
  • PERVs porcine endogenous retroviruses
  • porcine endothelial cells treated with ⁇ -galactosidase are capable of regenerating Gal epitopes on the cell surface within a few hours (LaVecchio, J.A., et al. Transplantation 60: 841-847, 1995).
  • pRBCs have a relatively short half-life in vivo.
  • any form of immunosuppressive therapy e.g. anti-CD 154 mAb therapy
  • the treatment would only be required temporarily.
  • Double Digestion of pRBCs with ⁇ -Galactosidase and Neuraminidase pRBCs are incubated with 100U of ⁇ -galactosidase per ml of pRBCs in PBS, pH6.0, containing polyethlene glycol. After incubating at 37°C for 4 hours, the cells are extensively washed with PBS. In order to avoid the potential problems associated with polyethlene glycol, a procedure using phosphate citrate buffer, ph 5.5 at 26°C for the enzyme treatment, has been developed. The pRBCs thus generated are deprived of Gal epitopes and are physiologically viable.
  • ⁇ -galactosidase-treated pRBCs are further digested with neuraminidase (1-2 units per ml of RBCs). After incubating for 2 hrs at 37°C with gentle rotation, pRBCs are then washed four times with PBS buffer.
  • the enzyme treatment of pRBCs can be monitored by flow cytometry analysis using purified anti-Gal and anti-NeuGc antibodies. After double digestion with both exoglycosidases, the resultant pRBCs are essentially non-reactive with either of the two preformed xenoreactive antibodies (anti- Gal and anti-NeuGc) present in human blood.
  • Example 2 Masking neuraminidase-treated pRBCs with NeuAc
  • asialyl-RBCs are likely to be unstable in vivo primarily due to the loss of negatively-charged residues from the cell surface and exposure of underlying carbohydrate structures.
  • ⁇ -galactosidase-treated pRBCs are recognized by anti-nonGal antibodies, but not by anti-Gal antibodies, it was possible to assess the effect of anti-nonGal antibodies on complement- mediated hemolysis by using enzyme-treated pRBCs in the assay, ⁇ - galactosidase-treated pRBCs underwent serum dosage-dependent hemolysis similar to the untreated pRBCs (above), although substantially more serum was required to achieve the same effect.
  • the L 50 value for the enzyme- treated pRBCs was 60-70 ⁇ l (in contrast to that of the untreated cells of 9-10 ⁇ l). Therefore, the data suggest that although anti-Gal antibodies in human serum account for the majority of complement-mediated hemolysis of pRBCs, anti-nonGal antibodies contribute significantly to this process.
  • Example 4 Identification of NeuGc as the major nonGal target on pRBCs.
  • N-glycolylneuraminic acid and N-acetylneuraminic acid (NeuAc) are two of the most abundant forms of sialic acid identified in glycoconjugates (Schauer, R. Sialic Acids. Chemistry. Metabolism, and Function. Wien: Springer, 1982). NeuGc is present in most animals, with the notable exception of humans and chickens (Asaoka, H., & Matsuda, H. J Vet Med Sci 56: 375-377, 1994).
  • the total cell membrane proteins which carry significant numbers of Gal and NeuGc epitopes, were isolated from pRBCs according to the published procedure (Zhu, A. Transplantation. 69, 2422-2428, 2000).
  • the membrane proteins were digested with ⁇ -galactosidase and neuraminidase under similar conditions to those described above.
  • the pre- and post- digestion protein samples were analyzed by immunoblotting using purified anti-Gal and anti-NeuGc as primary antibodies.
  • RNA was isolated from freshly frozen pig bone marrow according to a standard procedure.
  • the cDNA was then reverse-transcribed from the total RNA using a random primer, followed by PCR amplification using specific primers, hp-3 and hp-9 ( Figure 1).
  • An agarose gel analysis of the PCR product revealed a DNA fragment of approximately 1.4 kb. The fragment was isolated and subcloned into a PCR vector. Sequencing of the insert confirmed that the 1.4 kb fragment was the 3 '-end of the cDNA encoding procine CMP-NeuAc hydroxylase.
  • This 1.4 kb DNA fragment was radioactively labeled as a probe for screening a pig genomic library. After screening over one million plaques, more than ten possible 'positives' were selected using a duplicate- hybridization technique. After the second round of screening under identical conditions, three individual plaques were identified (# 41, 46 and 52).
  • lambda DNA was prepared and restriction digested for a Southern blot.
  • the probe for the Southern blot was the same as the one used for screening the library. All three clones generated DNA fragments strongly hybridized with the probe. Multiple bands on the Southern blot usually result from the internal restriction site of the fragment recognized by the probe.
  • different regions of the DNA were amplified using the long PCR procedure of Epicentre Technologies (Madison, WI). As shown in Figure 2, three overlapped fragments, 3 kb, 5 kb, and 9kb, were obtained using both universal primers (SP6 and T7) and specific primers. Based on the sequence data, there are at least three introns in the coding regions as indicated by the gray areas in Figure 2.
  • the sequence data is illustrated in Figure 3, wherein the shaded areas represent exons, totaling 224 residues. Shaded areas 1 through 5 are represented by SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.
  • the exons (SEQ ID NO:4 through SEQ ID NO: 8) have been combined to generate the comparison shown in Figure 4, wherein the porcine CMP-NeuAc hydrozylase is compared to the chimpanzee enzyme (SEQ ID NO:9) at the amino acid level.
  • the sequenced nucleotide regions of the porcine CMP-NeuAc hydrozylase, A-C correspond to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, respectively. The rest of the sequencing is routine.

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Abstract

L'invention concerne au moins deux antigènes importants présents dans les cellules d'espèces animales, telles que les porcs, ces antigènes provoquant une réponse immunitaire ou inflammatoire immédiatement après une implantation chez des êtres humains ou suite à un contact avec du sérum humain. Le premier antigène est un épitope α-galactosyl (Gal), par exemple, Galα(1->3)Galβ(1->4)GlcNac(linéaire B type 2) ou Galα(1->3)Galβ(1->4)Glc(linéaire B type 6). Le second antigène est une structure d'acide N-glycolylneuraminique (NeuGc). L'élimination de ces épitopes, de préférence par modification génétique de l'animal, de telle manière que l'épitope n'est pas produit ou est considérablement réduit ou par traitement chimique ou enzymatique des cellules animales, permet d'engendrer des organes, des tissus et des cellules appropriés à la xénotransplantation chez des êtres humains. On peut rendre ces cellules encore plus compatibles en modifiant génétiquement l'animal, afin qu'il exprime une protéine régulatrice humaine du complément (inhibitrice), telle que CD59, dans ses cellules ou qu'il exprime un excès d'une protéine régulatrice du complément chez le porc.
PCT/US2002/013564 2001-04-30 2002-04-29 Cellules et organes modifies destines a la xenotransplantation Ceased WO2002088351A1 (fr)

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